Rejection remains a potent threat to the long term success of organ transplants and significant morbidity is associated with even the best of modern immunosuppressive agents. The best hope for improving allograft survival, and particularly for preventing chronic rejection, is the induction of a state of donor-specific tolerance. The overall goal of this project is to explore the mechanism(s) of allograft tolerance which follows inoculation of the donor alloantigen directly into the thymus. Previous studies by ourselves and others have confirmed the efficacy of intrathymic inocula of endocrine or lymphohematopoietic cells to promote acceptance of subsequent extrathymic cellular or organ allografts in rodents. The first specific aim of this project is to examine whether deletion is the primary mechanism responsible for thymus-mediated tolerance. By utilizing several murine models with well-defined alloantigenic expressions, we will employ immune repertoire analysis of Vbeta-bearing T cells to explore clonal deletion as the basis for intrathymic tolerance. The second specific aim of this application is to assess the roles of the counterregulatory cytokine network as the mechanism of active clonotypic suppression responsible for the induction of intrathymic tolerance. Novel MHC knockout mice will be utilized to assess the role of the host lymphoid cells to promote specific cytokine profiles important in the induction and maintenance of tolerance following intrathymic inoculation with donor alloantigen. In specific aim 3 we will utilize a preclinical model of SCID mice reconstituted with human lymphohematopoietic and thymus grafts to simulate the human immune system. SCID-hu constructs will be used to assess the ability of the human thymus to nurture autografts and allografts of pancreatic islets and to reverse experimental diabetes. The impact of the residence of allogeneic islets in the human thymus on the peripheral T cell repertoire will be assessed as well as the possible induction of specific tolerance to extrathymic islets from the same donor. Intrathymic inocula of allogeneic lymphoid cells will also be tested as a tolerogen to promote donor-specific unresponsiveness to extrathymic islet allografts. Collectively, the present application proposes to delineate the mechanism(s) of acquired thymus-mediated immune tolerance and to utilize this information to optimize its potential utility in man.